Zur Transkriptions- und Translationskontrolle des Gens für Transitionsprotein 2
Dissertation
zur Erlangung des Doktorgrads
der Mathematisch-Naturwissenschaflichen Fakultäten der Georg-August-Universität zu Göttingen
vorgelegt von Özlem Topaloglu aus Istanbul/Türkei
Göttingen 2001
D7
Referent: Prof. Dr. W. Engel
Korreferent: Prof. Dr. U. Grossbach Tag der mündlichen Prüfung: 03.05.2001
CONTENTS
Page
ABBREVIATIONS VI
1. INTRODUCTION 1
2. MATERIALS AND METHODS 6
2.1 Materials 6
2.1.1 Chemicals 6
2.1.2 Solutions and buffers 7
2.1.3 Sterilization of solutions and equipments 8
2.1.4 Bacterial strains 8
2.1.5 Plasmids 9
2.1.6. Synthetic oligonucleotide primers 9
2.1.7 Antibodies 9
2.1.8 Animals 10
2.2 Methods 10
2.2.1 Isolation of nucleic acids 10
2.2.1.1 Isolation of genomic DNA from tissue samples 10
2.2.1.2 Isolation of total RNA from tissue 10
2.2.1.3 Isolation of poly(A)-enriched RNA 11
2.2.1.4 Isolation of plasmid DNA 11
2.2.1.4.1 Small-scale isolation of plasmid DNA 11
2.2.1.4.2 Large-scale isolation of plasmid DNA 12
2.2.1.4.3 Isolation of DNA fragments after agarose gel electrophoresis 13 2.2.1.4.4 Isolation of DNA fragments from acrylamide gels 13
2.2.2 Determination of the nucleic acid concentration 13
2.2.3 Enzymatic modification of DNA 14
2.2.3.1 Restriction of DNA 14
2.2.3.2 Dephosphorylation of 5‘ ends of DNA 14
2.2.3.3 Ligation of DNA fragments 15
2.2.3.4 TA-Cloning 15
2.2.3.5 Filling-up reaction 16
2.2.4 Gel electrophoresis 16
2.2.4.1 Agarose gel electrophoresis of DNA 16
2.2.4.2 Agarose gel electrophoresis of RNA 16
2.2.4.3 Polyacrylamide gel electrophoresis (PAGE)
of DNA fragments 17
2.2.4.4 SDS-PAGE for the separation of proteins 18 2.2.4.5 Acid-Urea gel electrophoresis for the separation of
small proteins 19
2.2.5 Labelling of nucleic acids 20
2.2.5.1 ”Random Prime” method for generation of
32P labeled DNA 20
2.2.5.2 5‘ End-labelling of oligonucleotides 20
2.2.5.3 Labelling of in vitro transcripts 20
2.2.6 Non-radioactive in vitro transcription 21 2.2.7 Non-radioactive dye terminator cycle sequencing 22
2.2.8 Blotting techniques 22
2.2.8.1 Dot blotting of DNA onto nitrocellulose filters 22 2.2.8.2 Southern blotting of DNA onto nitrocellulose filters 23 2.2.8.3 Northern blotting of RNA onto nitrocellulose filters 23 2.2.8.4 Western blotting of proteins onto nitrocellulose filters 24
2.2.9 Hybridization of nucleic acids 24
2.2.10 Isolation of proteins 25 2.2.10.1 Isolation of total proteins for CAT assay 25
2.2.10.2 Isolation of nuclear proteins 25
2.2.10.3 Isolation of nuclear basic proteins 26 2.2.10.4 Isolation of cytoplasmic S-100 proteins 26
2.2.11 Determination of protein concentration 27
2.2.12 Reverse transcriptase PCR (RT-PCR) 28
2.2.20 Non-radioactive CAT assay 29
2.2.21 Primer extension analysis 29
2.2.21.1 Labelling of the primer 29
2.2.21.2 Annealing of the primer to RNA 30
2.2.21.3 Primer extension reaction 30
2.22 Gel electrophoresis 31
2.23 Analysis of DNA-Protein interaction 31 2.23.1 Electrophoretic mobility shift assay (EMSA) 31
2.23.2 Southwestern analysis 32
2.24 Analysis of RNA-Protein interaction 32
2.24.1 RNA-Affinity chromatography 32
2.24.2 Northwestern analysis 34
2.25 Immunodetection 34
2.26. Histological techniques 35
2.26.1. Tissue Preparation for transmission electron microscopy (TEM) 35 2.26.2. Tissue Preparation for paraffin-embedding 35 2.26.3. Peroxidase anti-peroxidase technique (PAP) 36
3. RESULTS
3.1 Transcriptional regulation of rat Tnp2 gene 37
3.1.1 Primer extension analysis 37 3.1.2 Generation of Tnp2-147 transgenic mice 38 3.1.2.1 Plasmid construction for the transgenic mice 38 3.1.2.2 Genomic integration of the transgene 40 3.1.2.3 The testis-specific expression of the transgene 41 3.1.2.4 The testis-specific expression of transgenic CAT protein 42 3.1.2.5 The expression of the transgene during testis development 42 3.1.2.6 The expression of transgenic CAT protein during testis development 44
3.2 Translational regulation of rat Tnp2 mRNA 45
3.2.1 In vivo studies 46
3.2.1.1 Generation of Tnp2-SV40 transgenic mice line 46 3.2.1.1.1 Plasmid construction for the transgenic mice 46 3.2.1.1.2 Tissue-specific expression of the transgene 46 3.2.1.1.3 Tissue-specific expression of the transgenic CAT protein 47
3.2.1.2 Generation of Tnp2-hGH transgenic mouse line 48 3.2.1.2.1 Plasmid construction for the transgenic mice 48
3.2.1.2.2 Genomic integration of the transgene 50
3.2.1.2.3 Tissue-specific expression of the transgene 51 3.2.1.2.4 The expression of transgene during testis development 52 3.2.1.2.5 The expression of endogenous Tnp2 mRNA during
testis development 54
3.2.1.2.6 The expression of transgenic Tnp2 protein during
testis development 56
3.2.1.2.7 The expression of endogenous Tnp2 protein during
testis development 57
3.2.1.2.8 Immunohistochemical localization of transgenic Tnp2 protein 58 3.2.1.2.9 Electron microscopy of adult testis from Tnp2-hGH transgenic mouse 60
3.2.2 In vitro studies 61
3.2.2.1 Secondary structure prediction for 3’ UTR of Tnp2 mRNA 61
3.2.2.2 RNA-Affinity chromatography 62
3.2.2.3 Northwestern blot analysis 63
4. DISCUSSION
4.1 Transcriptional regulation of rat Tnp2 gene 65
4.2 Translational regulation of rat Tnp2 gene 69
4.2.1 Tnp2-SV40 transgenic mouse line 69
4.2.2. Tnp2-hGH transgenic mouse line 70
4.2.3 RNA-affinity chromatography 73
5. SUMMARY 75
6. REFERENCES 77
ABBREVIATIONS
BCP 1-bromo-3-chloropropane
BODIPY difluoride fluorophore (borondipyrromethane) BSA Bovine serum albumin
CAT Chloramphenical acetyltransferase CBB Coomasie brilliant blue
cpm counts per minutes DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid Dnase deoxyribonuclease DNTP deoxynucleotide DTT Dithiothreitol
EDTA Ethylene diamine tetraacetic acid
HEPES N-2-hydroxyethyl piperazine N'-2-ethane sulphonic acid IPTG Isopropyl-ß-thiogalactopyranoside
NaAc Sodium acetate
NBT Nitro-blue tetrazolium NTP Nucleotide
PCR Polymerase chain reaction PMSF Phenylmethylsulfonyl fuoride RNA Ribonucleic acid
RNase Ribonuclease
SDS Sodium dodecylsulfate SV 40 Simian Virus 40
TEMED Tetramethylethylene diamine TLC Thin layer chromatograhy
UV Ultra violet
X-Gal 5-bromo-4-chloro-3-indolyl-ß-galactosidase
1. INTRODUCTION
Spermatogenesis is the sequence of cytological events that result in the formation of highly specialized spermatozoa from undifferentiated stem cells, namely spermatogonia (Bellve, 1979). This process takes place within the seminiferous tubule throughout the reproductive life-span of the male. Spermatogenesis can be divided into four distinct phases: (1) the proliferation and renewal of undifferentiated spermatogonia; (2) the differentiation of spermatogonia; (3) the process of meiosis; (4) spermatid development (spermiogenesis) and release of spermatozoa (spermiation). During the final step which is called spermiogenesis, round spermatids undergo several morphological, biochemical and physiological modifications which result in the formation of mature spermatozoa. These include the nuclear reorganization, the development of the acrosomic system originating from the Golgi apparatus, the assembly of tail structures and cytoplasmic reorganization whose final phase results in the release of spermatozoa in the lumen of the seminiferous tubules (Daduone et al., 1993; Russel et al., 1990). The sequence of morphological events has been divided into different successive steps which vary in number with the species: 8 in man, 10 in the baboon, 15 in the ram, bull and boar, 16 in the mouse and 19 in the rat (Oakberg, 1956).
Nuclear reorganization in haploid spermatids involves the change of nucleosomal chromatin to the highly condensed chromatin found in the sperm nucleus. In mammals, early spermatids, which have nucleosomal chromatin, contain a mixture of somatic histones as well as testis- specific variants of H1 and H2B and sometimes H2A and H3 (Meistrich, 1989). About midway through spermatid development, the nucleus begins to condense, to elongate, and to become resistant to mechanical disruption. These nuclear changes coincide with the replacement of both the somatic-type and testis-specific histones with a set of spermatid- specific chromosomal proteins which are referred to as „transition proteins“. Transition proteins are small lysine- and arginine-rich proteins that play role in the transformation of the nucleosomal chromatin into smooth condensed chromatin fibers. In the final stages of nuclear restructuring, the transition proteins are replaced by the distinctive cysteine-rich protamines to produce the tightly compacted nucleus of the spermatozoon (Balhorn et al., 1984; Balhorn, 1989; Bellve et al., 1983; Dadoune et al., 1993).
During spermatogenesis, stringent temporal and stage-specific gene expression is a prerequisite for the correct differentiation of male germ cells. There are two levels of transcriptional regulation: Methylation and binding of the trans-acting factors to the TATA- box, and other specific DNA sequences in the promoter region of nucleoproteins (Goldberg, 1996). The dogma which dictates that methylation of specific cytosines in DNA leads to a reduction or cessation of gene transcription is not valid for all spermatid-specific genes. The opposite occurs in the chromosomal locus on mouse chromosome 16, which contains the protamine 1 and 2, transition protein 2 genes which are located in a large methylated region in their expressing cell type, namely round spermatids (Choi et al., 1997). On the other hand, the gene for transition protein 1 reveals a cell-specific demethylation associated with gene activity (Trasler et al., 1990). Another evidence that general transcription may be differentially regulated in germ cells is the accumulation of the TATA-binding protein (TBP) in early haploid germ cells at much higher levels than in any other somatic cell types. It has been calculated that adult testis contains 7.9 and 11.4 fold more molecules of TBP per haploid genome equivalent than adult spleen and liver cells, respectively (Schmidt and Schibler, 1997). Additionally, TFIIB and RNA polymerase II were also found to be overexpressed in testis (Schmidt and Schibler, 1995).
Studies in transgenic mice have shown that relatively short 5‘ upstream sequences can direct tissue- and stage-specific expression of haploid expressed genes in testis (Howard et al., 1993;
Peschon et al., 1987; 1989; Reddi et al., 1999). Zambrowicz et al. have demonstrated that a 113 bp region from –150 to –37 of the mouse protamine 1 can successfully direct spermatid- specific transcription in transgenic mice (Zambrowicz et al., 1993). A number of ubiquitous and testis-specific proteins bind to this region (Zambrowicz et al., 1994), including Tet-1 which is a testis-specific trans-acting nuclear protein that recognizes the 11-mer sequence at – 64 in 5‘UTR of mouse protamine 1 (Tamura et al., 1992). Analysis of the mouse protamine 2 promoter by in vitro transcription assays have identified a potential positive regulatory region from –140 to –23 (Bunick et al., 1990). Mobility shift assays revealed binding of both ubiquitious and testis-specific proteins (Johnsons et al., 1991). Furthermore, there is evidence of binding for a novel orphan nuclear factor and CRE at positions –64/-48 and –84/-72, respectively (Enmark and Gustafsson, 1996; Delmas et al., 1993). The cAMP response element (CRE)-like sequences have been found in a number of genes that are transcribed in the spermatids. Expression of cAMP response element modulator (CREMτ) appears to be restricted to the testis. There is direct evidence that CREMτ is one of the transcription factors responsible for the postmeiotic expression of the calspermine gene (Sun et al., 1995) and the
transition protein 1 gene (Kistler et al., 1994). In two recent reports (Blendy et al., 1996 and Nantel et al., 1996) the specific role of CREM in spermiogenesis was addressed using CREM-mutant mice which showed postmeiotic arrrest at the first stage of of spermiogenesis.
Absence of the CREM gene leads to the lack of expression of CREM-dependent genes such as genes for protamine 1 and 2, and calspermin.
Post-transcriptional control is especially important towards the end of spermatogenesis since the global transcription ceases several days before the completion of spermiogenesis. Thus, mRNA storage and translational activation play important roles in the expression of many spermatid and spermatozoon proteins which are synthesized in late stages of germ cell maturation. For example, although the genes for transition proteins and protamines are transcribed in round and elongating spermatids, their mRNAs are stored as ribonucleoprotein (RNP) particles in a translationally repressed state for several days until they are translated in elongating and elongated spermatids (Dadoune et al., 1995; Eddy et al., 1993; Hecht et al., 1989; 1990a; 1990b; Morales et al., 1991; Kleene et al., 1996). The need for the translational regulation was demonstrated in transgenic mice where the mouse protamine 1 mRNA was prematurely translated by exchanging the 3'UTR of protamine 1 with the 3'UTR of human growth hormone (hGH). Premature accumulation of Prm1 mRNA resulted in dominant male sterility accompanied by a complete arrest in spermatid differentiation, early condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein in transgenic mice (Lee et al., 1995). Deadenylation and the interaction between RNA-binding proteins and both/either 5' untranslated regions and/or 3' untranslated regions are the main suggested mechanisms to achieve the translational regulation of testis-specific genes. It was shown that the length of poly-A tails of transition proteins and protamine mRNAs correlates with translational activity (Heidaran and Kistler, 1987; Kleene et al., 1984;
Kleene, 1989; 1993). The mRNAs for protamines are subjected to a shortening process before or during translation, namely from 0.62 kb to 0.45 kb for protamine 1 and from 0.9 kb to 0.7 for protamine 2 (Domenjoud et al., 1991). This reduction in poly (A) length of mRNAs appears restricted to the haploid cells of the testis, because mRNAs expressed in premeiotic or meiotic male germ cells encoding ornithine decarboxylase, lactate dehydrogenase c, and cytochrome c show increases in polyadenylation (Hecht, 1998). One of the best characterized RNA-binding protein is the 70 kDa poly-A binding protein (PABP) whose mRNA level increases as germ cells enter meiosis (Gu et al., 1995). The high level of PABP in round spermatids and in mRNPs suggests that it may have a role in the storage of developmentally regulated mRNAs in mammalian testis. It has been suggested that PABP migrates from the
poly (A) tail to the AU-rich segments in the 3'UTR, leaving the poly (A) tail naked and vulnerable to degradation (Bernstein and Ross, 1989).
Translational repression in round spermatids is achieved by the binding of sequence-specific RNA-binding proteins to the 3'UTRs, whereas translation in elongating spermatids is achieved by the covalent modification of the mRNP complex and release of translatable protamine 1 mRNA (Braun, 1990). The studies of Braun et al. have demonstrated that the 3' UTR of mouse protamine 1 gene contains all of the elements required for proper translational regulation of protamine 1 mRNA in transgenic mice (Braun et al., 1989). Subsequent studies identified cis-acting elements in the 3'UTRs of protamine 1 and 2 mRNAs and trans-acting factors that recognize them. All of the cis-acting sequences required for the translational regulation of protamine 1 has been mapped to 62 nucleotides in the 3' most region of the protamine 1 3' UTR (Fajardo et al., 1997). A growing number of RNA-binding proteins that influence the translation through binding to the 3'UTR of mouse protamine 1 and 2 mRNAs have been identified with the help of gel mobility shift assays and UV-crosslinking experiments. Screening of male germ cell cDNA expression libraries with 3' UTR has also yielded several clones encoding RNA-binding proteins like protamine 1 RNA-binding protein (Prbp) which was later shown to be required for the activation of repressed protamine 1 mRNA (Lee et al.,1996; Zhong et al., 1999), testis-brain-RNA-binding protein (TB-RBP) which supresses the translation of protamine 1 and 2 in vitro and attaches mRNAs to microtubules by binding to conserved elements in the 3' untranslated regions of specific mRNAs, spermatid perinuclear RNA binding protein (Spnr) which is highly expressed in elongating haploid germ cells, is localized to a spermatid-specific microtubule array called the manchette and thereby might play a role in the putative subcellular localization of protamine mRNA molecules that are destined to be activated for translation at the nuclear periphery (Schumacher et al, 1995), and finally Tenr which is an RNA-binding protein, is localized in a lattice-like network within the spermatid nucleus (Schumacher et al., 1995).In this study, the transcriptional and translational regulation of rat transition protein 2 (Tnp2) were investigated.
Transition proteins are quite variable with regard to size and amino acid compositions. They are generally more basic than histones but less basic than protamines. They appear to be species- or perhaps class-specific proteins. In mammals, during elongation and condensation of the spermatid nucleus, several transition proteins have been characterized. In boar, man, mouse, ram and rat, this family consists of four proteins, Tnp1-4, of which Tnp1 and Tnp2 are best characterized. Tnp2 is about the molecular size of a core histone and is characterized by a large amount of basic residues (~32%), serine (~22%) and proline (~13%) and by the
presence of cysteine (5%) (Wouters-Tyrou et al., 1989). Tnp2 consists of 115 amino acids in rat and has characteristic domains (Luerssen et al., 1989). The carboxyl terminal is enriched in basic residues and is likely to be the major site of DNA-binding (Cole et al., 1987). The amino terminal region binds zinc and has two proposed zinc finger-like structures (Kundu et al., 1994). The transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14 in mouse (Shih and Kleene, 1992). On the other hand, the Tnp2 protein can be firstly detected in step 12 spermatids, is strongly present in step13, and finally disappears in step 14 spermatids in the mouse (Alfons and Kistler, 1993).
Aim of the study
In the framework of this study, the transcriptional and translational regulation of rat Tnp2 gene was to be investigated by means of both in vitro and in vivo systems. The rat Tnp2 promoter region should be characterized by primer extension analysis and analysis of transgenic promoter/reporter strains. Based on prior information of a transgenic line, carrying 525 bp 5’ untranslated region, the relevant region should be narrowed down by further transgenic mice. The role of 3’UTR in translational repression of Tnp2 mRNA should be investigated by further transgenic lines. In analogy to a reported mouse protamine 1 transgene, where the 3' UTR of protamine was replaced by human growth hormone 3' UTR, a similar transgenic line for a precocious Tnp2 expressing transgenic strain was planned. By RNA-affinity chromatography RNA-binding proteins that are specifically bind to the Tnp2 3’
UTR mRNA was planned to be enriched, isolated, and cloned.
2. MATERIALS AND METHODS
2.1 Materials
2.1.1 Chemicals
Acetyl CoA Sigma, Deisenhofen
Acrylamide Gibco/BRL, Eggstein
Ammonium sulfate Sigma, Deisenhofen
Aprotinin Sigma, Deisenhofen
Agar Difco, Detroit, USA
Agarose Gibco/BRL, Eggstein
Ampicillin Sigma, Deisenhofen
Aprotinin Sigma, Deisenhofen
BSA Biomol, Hamburg
CAM Substrate Strategene, Heidelberg
Choloroform Roth, Karlsruhe
Dextran sulfate Pharmacia, Freiburg
Dithiothreitol Biomol, Hamburg
DNA Markers GibcoBRL, MBI
DNA Ligase GibcoBRL, MBI
Dnase Boehringer, Mannheim
dNTPs Boehringer, Mannheim
Dye Terminator Mix Applied Biosystems Ethidium bromide Sigma, Deisenhofen
Formamide Merck
Ethanol Roth, Karlsruhe
Glycine Biomol, Hamburg
JETPREP-Plasmid Midi Kit Genomed, Bad Oeynhausen
Pepstatin Sigma, Deisenhofen
Phenol Biomol, Hamburg
Picric acid Fluka, Neu Ulm Poly (dI.dC)(dI.dC) ICN, Cleveland, USA PMSF (Phenylmethylsulfonylfluoride) Sigma, Deisenhofen
Proteinase K Boehringer; Mannheim
Protein marker Biorad, Sigma
SDS Serva, Heidelberg
Spermidin Sigma, Deisenhofen
Urea ICN, Cleveland, USA
Triton X-100 Serva; Heidelberg
tRNA Boehringer; Mannheim
Tween-20 Fluka; Neu Ulm
X-Gal Boehringer; Mannheim
2.1.2 Solutions and buffers
Ampicillin 50 ug/mL H2O
Denaturing Solution 1.5 M NaCl
0.5 M NaOH
Denhardt Solution (50x) 1 % BSA
1 % Polyvinylpyrrolidone
1 % Ficoll 400
E-Buffer (10x) 300 mM NaH2PO4 pH 7.0 50 mM EDTA
Hybridization Solution 12.5 % Denhardt Solution
5 % Dextran sulfate
0.25 % SDS
5 % SSC
LB-Agar pH 7.2 LB- Medium with
1.5 % Bacto-Agar
LB-Medium pH 7.2 1 % Bacto-Tryptone
0.5 % Bacto-Yeast-Extract
1 % NaCl
Sodium acetate 3 M Sodium acetate pH 5.3
Neutralization solution 1 M Tris pH 5.5 3 M NaCl
SSC (20x) 0.3 M Tri-sodium acetate pH 7.0
3 M NaCl
TBE Buffer (5x) 225 mM Tris pH 8.3
225 mM Boric acid
10 mM EDTA
TE Buffer (10x) 100 mM Tris pH 8.0
10 mM EDTA
2.1.3 Sterilization of solutions and equipments
All solutions, which are not heat sensitive, were sterilized at 121°C, 105 Pa for 60 min in an autoclave (Webeco, Bad Schwartau). Heat sensitive solutions were filtered through a disposable sterile filter (0.2 to 0.45 µm pore size). Plasticware was autoclaved, as above.
Glassware was sterilized overnight in an oven at 220°C.
2.1.4 Bacterial strains
E.coli HB 101 (Boliver and Beckman, 1979) E.coli JM 109 (Messing, 1983)
E.coli XL1 Blue (Young and Davis, 1983)
2.1.5 Plasmids
Bluescript II SK (-) Stratagene, La Jolla, USA
pCAT3 Promega, Wisconsin, USA
pGEM-T, pGEM-T EASY Promega, Wisconsin, USA
2.1.6 Synthetic oligonucleotide primers
The synthetic oligonucletide primers used in this study were ordered from either NAPS (Göttingen, Germany) or Roth (Karlsruhe, Germany).
Tppro1-F 5‘ GGG GCG AGC TCT GCC ATA CCT GTC ACC 3‘
RTP2-5A 5‘ CAT GGG ATC CCA CAC TCG GAG GG 3‘
RTP2-PE1 5‘ GTG GTG GCT GCA GGC ACA CTG 3‘
RTP2-KpnI-F 5‘ GAA GGT ACC AAG TGA CAC AC 3‘
Tp2-E2-R 5‘ GGA ATT CTC ACT TGT ATC TTC GTC C 3‘
hGH-3‘UTR-F 5‘ GGA ATT CCT GCC CGG GTG GCA TCC C 3‘
hGH-3‘UTR-R 5‘ CCC AAG CTT CAA ACC ACC CCC CTC CAC 3‘
EcoRI-(T)30-R 5‘ CGG AAT TCT TTT TTT TTT TTT TTT TTT TTT
TTT TTT TTC CAG GGT ACA AGC 3‘
Tp2-Ex1-F 5‘ GAC ACC AAG ATG CAG AGC CTT 3‘
2.1.7 Antibodies
For the detection of chloramphenicol acetyltransferase (CAT) protein in Tnp2-147 transgenic line, the polyclonal rabbit anti-CAT antibody from BioTrend, Köln, Germany was used.
The polyclonal rabbit anti-Tnp2 antibody was kindly provided by S.K. Kister, University of South Carolina, Columbia, USA (Alfons and Kistler, 1993).
To carry out immunodetection in hGH-Tnp2 transgenic line, the rabbit peroxidase-anti- peroxidase complex from Dako, Hamburg, Germany was used.
2.1.8 Animals
In this study NMRI and CD-1 mice lines were used for both RNA and protein preparations which are bred at Institute of Human Genetics, Göttingen, Germany.
SIV 50 rat line was utilized for the preparation of nuclear and cytoplasmic proteins from rat.
2.2 Methods
2.2.1 Isolation of nucleic acids
2.2.1.1 Isolation of genomic DNA from tissue samples
Lysis Buffer 50 mM Tris-HCl pH 8.0
100 mM EDTA
0.5 % SDS
The method employed was the same as that of Laird et al. (1991). 1 to 2 cm of the tail from a mouse was incubated in 700 µl of lysis buffer containing 35 µl proteinase K (10 ug/ul) at 55°C overnight. Equal volume of phenol was added, mixed by inverting, and centrifuged at 8000 xg at room temperature for 10 min. After transferring the aqueous layer into a new tube, the same procedure was repeated, but this time with 0.5 Vol phenol and 0.5 Vol chloroform.
Then, the DNA was precipitated with 2.5 Vol 100 % ethanol, and fished-out with a pipette tip.
Usually, it was dissolved in 100-200 µl of 1 x TE buffer.
2.2.1.2 Isolation of total RNA from tissue
TRI Reagent is an improved version of the single-step method for total RNA isolation. The composition of TRI Reagent includes phenol and guanidine thiocyanate in a mono-phase solution.
100-200 mg tissue sample was homogenized in 1-2 ml of TRI Reagent by using a glass-teflon homogenizer. The sample volume should not exceed 10 % of the volume of TRI Reagent used for the homogenization. The homogenate was incubated at room temperature for 5 min to
permit the complete dissociation of nucleoprotein complexes. Then, 0.1-0.2 ml of BCP was added which is a less toxic chemical than chloroform, shaked vigorously, and stored at room temperature for 15 minutes. After centrifugating the sample at 12000 xg for 15 min at 4°C, the colorless upper aqueous phase was transferred into a new tube. The RNA was precipitated by adding 0.5 ml of isopropanol. Finally, the pellet was washed with 75 % ethanol, and dissolved in 80-100 µl of DEPC-H2O.
2.2.1.3 Isolation of poly(A)-enriched RNA
To isolate polyadenylated mRNA, the Qiagen Oligotex kit was employed. The purification procedure makes use of oligo-dT coated latex particles that provide a hybridization carrier on which nucleic acids containing polyadenylic acid sequences can efficiently immobilized and easily recovered.
400 µg total RNA was mixed with 400 µl of 2x binding buffer and 30 µl of oligotex suspension and incubated for 3 minutes at 65°C to distrupt the secondary structure of the RNA. It was then further incubated for 10 min at room temperature to allow the hybridization between the oligo (dT)30 linked to the latex particles and the poly (A) tail of the mRNA. The oligotex resin containing the RNA was pelleted by centrifugation for 2 min at full speed.
After removing the supernatant, the pellet was resuspended in 400 µl of wash buffer QW2, then transferred to a spin column. The column was washed 2 times with wash buffer QW2, and finally the RNA was eluted with 100 µl of H2O.
2.2.1.4 Isolation of plasmid DNA
2.2.1.4.1 Small-scale isolation of plasmid DNA
5 ml of LB medium with the appropriate antibiotic was inoculated with a single E. coli colony and incubated overnight at 37°C with shaking. 1.5 ml of this culture was centriguted at 5000 xg for 10 minutes. The pellet was resuspended in 200 µl of solution E1. After adding equal volumes of solutions E2 and E3, respectively, the pellet was incubated on ice for 15 minutes, and centrifuged at full speed at 4°C. The supernatant was transferred into a new tube, and 1 ml of 100 % ethanol was added to precipitate the DNA. It was then stored at –20°C for 30
minutes, centrifuged at full speed for 30 minutes, and finally the pellet was dissolved in 30 µl of H2O.
2.2.1.4.2 Large-Scale isolation of plasmid DNA
80 ml of LB medium was inoculated with a single E.coli colony and incubated overnight at 37°C with shaking. In order to pellet the cells, it was centrifuged at 5000 rpm for 10 min. The pellet was resuspended in 4 ml of solution E1. To lyse the cells 4 ml of solution E2 was added, mixed gently, and incubated for 2-3 minutes at room temperature. Equal amount of solution E3 was added to the tube, and mixed immediately by inverting. The mixture was centrifuged at 4000 rpm for 30 minutes at 20°C. Meanwhile, the column that was provided by the kit was equilibrated with 10 ml of solution E4. The clear lysate after the centrifugation was applied to the equilibrated column. The column then was washed twice with 10 ml of solution E5. Finally, the DNA was eluted with 5 ml of solution E6. To precipitate the DNA, 0.7 Vol of isopropanol was added, and centrifuged at 4000 rpm for an hour at 12°C. The DNA was usually dissolved in 100 ul of H2O.
Solution E1 50 mM Tris/HCl pH 8.0 Solution E4 600 mM NaCl
10 mM EDTA 100 mM NaAc pH 5.0 100 µg/ml RNase 0.15 % Triton X-100
Solution E2 200 mM NaOH Solution E5 800 mM NaCl
1% SDS 100 mM NaAc pH 5.0
Solution E3 3.1 M Potassium acetate Solution E6 1250 mM NaCl pH 5.5 100 mM Tris/HCl
pH 8.5
2.2.1.4.3 Isolation of DNA fragments after agarose gel electrophoresis
For the isolation of DNA fragments which are 300-4000 bp in length from agarose gels, the Geneclean kit from Biomol 101 (Biomol, Hamburg) was employed. The principle of this method depends on the binding capacity of DNA to silica in high salt concentrations and elution in low salt solutions. After separation of DNA on an agarose gel, the DNA band to be isolated was excised with a razor blade, and weighed. 3 Vol of 6 M NaI was added to the tube, and the agarose slice was melted at 55°C. Depending on the DNA amount, required amount of GLASSMILK which is an aqueous suspension of silica matrix was added and the tube was placed on ice for 30 min. After centrifuging it at full speed for 2 min, the pellet was washed 2 times with „New Wash“, and allowed to dry at room temperature. To elute the DNA, the pellet was resuspended in 30 µl of H2O and incubated at room temperature for 10 min. After the final centrifugation at 14000 rpm for 5 min, the supernatant containing the DNA was transferred into a new tube.
2.2.1.4.4 Isolation of DNA fragments from acrylamide gels
For the isolation of small DNA fragments (50-300 bp), the DNA was first separated on a polyacrylamide gel. After staining the gel with ethidium bromide, the desired band was cut out and transferred into a 1.5 ml centrifuge tube. 500 µl TE buffer was added to the gel slice and incubated at 50°C overnight, with shaking. After centrifugation at 12000 xg at room temperature for 15 minutes, the supernatant containing DNA was precipitated by adding 1/10 Vol 3 M NaAc and 2.5 Vol 100 % of ethanol.
2.2.2 Determination of the nucleic acid concentration
The concentration of nucleic acids was determined photometrically by measuring absorption of the samples at 260 nm. DNA quality i.e. contamination with salt and protein was checked by the measurements at 230, 280, and 320 nm. The concentration can be calculated according to the formula:
C = (E 260 – E 320) x f x c
C = concentration of sample (ug/ul) E 260 = absorption at 260 nm
E 320 = absorption at 320 nm
f = dilution factor
c = concentration (standard) / absorption (standard) for double stranded DNA : c = 0.05 ug/ ul
for RNA : c = 0.04 ug/ul
for single stranded DNA : c = 0.03 ug/ul
2.2.3 Enzymatic modifications of DNA
2.2.3.1 Restriction of DNA
Restriction enzyme digestions were performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. Typical digestions include 2-10 U enzyme per microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions were usually incubated for 1-3 hrs to insure complete digestion at the optimal temperature for enzyme activity which was typically 37°C.
2.2.3.2 Dephosphorylation of 5‘ ends of DNA
To prevent the recircularization of vector plasmid without insertion of foreign DNA, alkaline phosphatase treatment was peformed. Alkaline phosphatase catalyses the hydrolysis of 5'- phosphate residues from DNA. The followings were mixed,
1-5 µg vector DNA
5 µl 10 x reaction buffer
1 µl alkaline phosphatase (1 U) in a total volume of 50 µl
and incubated at 37°C for 30 min. Then the reaction was stopped by heating at 85°C for 15 min.
2.2.3.3 Ligation of DNA fragments
The ligation of an insert into a vector was carried out in the following reaction mix:
30 ng vector DNA
50-100 ng insert DNA
1 µl ligation buffer (10 x) 1 µl T4 DNA ligase (5U/µl) in a total volume of 10 µl.
Blunt-end ligations were carried out at 16°C overnight whereas sticky-end ligations were carried out at room temperature for 2-4 hrs.
2.2.3.4 TA-Cloning
Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated addition of a single nucleotide to the 3'-ends of PCR products. In the presence of all 4 dNTPs, dA is preferentially added. This complicates cloning, as the supposedly blunt- ended PCR product often is not. This terminal transferase activity is the basis of the TA- cloning strategy. For the cloning of PCR products, pGEM-T Easy Vector system which has 5‘
T overhangs was employed. The followings were mixed,
50 ng of pGEM-T or pGEM-T Easy Vector PCR product ( 3:1 vector : insert ratio) 1 µl T4 DNA Ligase 10x buffer 1 µl T4 DNA Ligase
in a total volume of 10 µl
The contents were mixed by pipetting and the reaction was incubated overnight at 4°C. To transform the ligation reaction JM 109 competent cells were used.
2.2.3.5 Filling-up reaction
0.1-4 µg of digested DNA was mixed with 0.05 mM dNTPs and 1-5 U of Klenow fragment.
The reaction was incubated at 37°C for 10 min, then stopped by heating at 75°C for 10 min.
2.2.4 Gel electrophoresis
Gel electrophoresis is the technique by which mixtures of charged macromolecules, especially nucleic acids and proteins, are rapidly resolved in an electrical field.
2.2.4.1 Agarose gel electrohoresis of DNA
Agarose gels are used to electrophorese nucleic acid molecules from as small as 150 bases to more than 50 kilobases, depending on the concentration of the agarose and the precise nature of the applied electrical field (constant or pulse).
1 g of agarose was dissolved in 100 ml 0.5 x TBE buffer, and boiled in the microwave, then cooled down to about 50°C before adding 3 µl ethidium bromide (10 mg/ml). The gel was poured into a horizantal gel chamber.
2.2.4.2 Agarose gel electrophoresis of RNA
Single-stranded RNA molecules often have small regions that can form base-paired secondary structures. To prevent this, the RNA should be run on a denaturing agarose gel which contains formaldehyde, and additionally is pre-treated with formaldehyde and formamide.
1.25 g of agarose was dissolved in 100 ml of 1 x E-Buffer, after cooling it to about 50°C, 25 ml of formaldehyde (37 %) was added, stirred and poured into a vertical gel chamber. To 10-20 µg of RNA,
2 µl 10 x E-Buffer 3 µl Formaldehyde 8 µl Formamide (40%)
1.5 µl Ethidium bromide
were added and the samples were denatured at 65°C for 10 min, and chilled on ice before applying to the gel. The gel was run at 80 V at 4°C for about 3-4 hrs.
2.2.4.3 Polyacrylamide gel electrophoresis (PAGE) of DNA fragments
Polyacrylamide gel electrophoresis was employed to analyze the small DNA fragments which were 50-500 bp and to separate the gel retardation samples. The percentage of acrylamide (6- 12 % w/v) determines the resolving property of the gel. A 10 % of PAG was prepared as follows:
2.5 ml 40% PAA stock solution 2.5 ml 5 x TBE buffer
150 µl APS (10 % w/v)
15 µl TEMED
in a total volume of 10 µl
APS and TEMED were used to initiate the polymerization of the gel. The gel was poured vertically between two clean glass plates, ensuring no air bubbles. After completion of the electrophoresis, DNA was visualized by staining the gel in an ethidium bromide solution.
2.2.4.4 SDS-PAGE for the separation of proteins (Laemmli, 1970)
Sample buffer (2 x) 0.5 M Tris/HCl pH 6.8 20%Glycerol
4% SDS
10 % ß-Mercaptoethanol
Running buffer (5 x) 25 mM Tris/HCl pH 8.3 (Tris/Glycine buffer) 192 mM Glycine
0.1 % SDS
Stacking gel buffer (4 x) 0.5 M Tris/HCl pH 6.8
0.4 % SDS
Separating gel buffer (4 x) 1.5 M Tris/HCl pH 8.8
0.4 % SDS
SDS gel electrophoresis is a method for separating proteins within a sample for analysis and molecular weight detemination. The proteins are denatured and rendered monomeric by boiling in the presence of reducing agents (2-merceptoethanol or dithiotheitol) and negatively charged detergent (SDS).The proteins which normally differ according to their charges are all coated with the SDS which is negatively charged. Hence, all the proteins in the sample become negatively charged. In this way, the separation is according to the size of the proteins.
A SDS-PAG consists of two gels; firstly, usually 10 % separating gel was poured. In order to achieve a smooth boundary between separating and stacking gel, the separating gel was covered with a layer of water saturated butanol. After the polymerization of the separating gel completed, a 4 % stacking gel was poured. The samples were boiled in sample buffer for 10 minutes at 95°C before applying to the gel. The gel was run at 15 mA for one hour then at a constant current of 30 mA.
2.2.4.5 Acid-Urea gel electrophoresis for the separation of small proteins (Panyim and Chalkey, 1969)
This gel system is a SDS- free PAGE and applied specially for the separation of basic proteins which have small molecular weight like histones, transition protein 1 and 2 and protamines. In the absence of SDS, the proteins would still be separated essentially on the basis of their sizes, but their charges would vary according to their amino acid contents. This is because of the charge on a protein at any particular pH is the sum of the charges prevailing on the side chain groups of it s constituent amino acid residues, and the free amino and carboxyl groups at it s termini. Thus, in an ionic detergent-free gel electrophoretic system, both the molecular size and charge act as bases for effective protein separation. The pH prevailing in such a system might be anything, but it is commonly about pH 3.0. At pH 3.0, all proteins are likely to be positively charged and to travel towards the cathode in an electrical field. In an acid- polyacrylamide gel electrophoresis system, two proteins of similar size but different charge maybe separated from each other. In this work, this gel system was employed method for the separation of small basic proteins such as histones, transition protein 1 and 2, and protamines.
For this purpose, 15 % polyacrylamide with 2.5 M urea and 5 % acetic acid was prepared as follows:
3 g Urea
7.5 ml 40 % PAA 1 ml 100 % Acetic acid 75 µl TEMED
500 µl APS
in a total volume of 20 ml
The electrophoresis was carried out for 4 hours at 15 mA. The gel was run in the direction from anode to cathode. A stop mix containing 2.5 M urea, 5 % acetic acid, and 40 % saccharose was used.
2.2.5 Labelling of nucleic acids
2.2.5.1 „Random Prime“ method for generation of 32P labelled DNA (Feinberg and Vogelstein, 1989)
Ready-To-Go DNA labelling kit (Pharmacia) was employed for labelling of DNA fragments radioactively. The method relies on the random priming principle developed by Feinberg and Vogelstein. The reaction mix contains dATP, dGTP, dTTP, Klenow fragment (4-8 units) and random oligodeoxyribonucleotides, primarily 9-mers. 25-50 ng of DNA was denatured in a total volume of 46 µl at 95°C for 15 minutes. It is then transferred to Ready-To-Go reaction cup, mixed thoroughly by vortexing, and finally 4 µl of [α-32P] dCTP (3000 uCi/mmol) was added to the reaction mixture. The labelling reaction was carried out at 37°C for 1-3 hours.
2.2.5.2 5‘ End-Labelling of oligonucleotides
The oligonucleotides which were used in electrophoretic mobility shift assay were labelled as follows:
20-50 ng ds DNA
2 ul 10 x Buffer for Klenow Fragment 1 ul Klenow Fragment
3 ul [α-32P] dCTP
in a total volume of 20 ul
The labelling reaction was carried at room temperature for 10 min. Then, the labelled DNA was purified with the use of MicroSpin G-25 Columns (Amersham Pharmacia Biotech Inc., NJ, USA).
2.2.5.3 Labelling of in vitro transcripts
The RNA which was used in Northwestern analyses was in vitro transcribed from a lineralized vector in the presence of [α-32P] UTP.
2-5 µg lineralized plasmid 1 µl RNasin (20-40 U)
1 µl 10 mM ATP; CTP; GTP 1 µl 1 mM UTP
4 µl 5 x Transcription buffer 2 µl 0.1 M DTT
1 µl [α-32P] UTP (800 µCi/mMol; 16 µCi) 1 µl RNA Polymerase (T7, T3 or SP6) in a total volume of 20 µl
The contents were mixed, and incubated at 37°C for an hour. In order to remove the DNA template, 1 µl of RNase-free DNase I (1 U/µl) was added and incubated further for 15 min.
Finally, the in vitro transcribed RNA was precipitated by adding 1/10 Vol of 3 M NaAc and 2.5 Vol of 100 % ethanol, and dissolved in 100 µl of DEPC-H2O.
2.2.6 Non-radioactive in vitro transcription
Ambion MEGAshortscript kit was used to obtain high yields of in vitro transcription products in the 20-500 nucleotide range. These high yields were achieved by optimizing reaction conditions for RNA synthesis in the presence of high nucleotide and polymerase concentrations.
10 ug template DNA in a volume not to exceed 8 ul 2 µl 10x Transcription buffer
2 µl ATP solution (75 mM) 2 µl CTP solution (75 mM) 2 µl GTP solution (75 mM) 2 ul µl UTP solution (75 mM)
2 µl T7 MEGAshortscript enzyme mix -- µl RNase-free dH2O
in a 20 µl of total volume
The contents were mixed, and incubated at 37°C for 4 hrs. The removal of template DNA and the precipitation of the in vitro transcribed RNA were achieved as described in section 2.2.5.3.
2.2.7 Non-Radioactive dye terminator cycle sequencing
The non-radioactive sequencing was achieved with Dye Terminator Cycle Sequencing-Kit (ABI, Weiterstadt) and the reaction products were analyzed with automatic sequencing equipment, namely 373 A DNA Sequencer (ABI, Weiterstadt). For the sequencing reaction, four different dye labelled dideoxy nucleotides were used, which, when exposed to an argon laser, fluorescent emitting light which could be detected and interpreted. The reaction was carried in a total volume of 10 µl containing 1 µg plasmid DNA or 100-200 ng purified PCR products, 10 pmol primer and 4 µl reaction mix (contains dNTPs, dideoxy dye terminators and Taq DNA polymerase). Elongation and chain termination takes place during the following program in a thermocycler: 5 min denaturing followed by 25 cycles 95°C 30 sec, denaturing; 55°C 15 sec, annealing; 70°C 4 min, elongation. After the sequencing reaction, the DNA was precipitated with 1/10 Vol 3 M NaAc and 2.5 Vol 100 % ethanol. The pellet was dissolved in 4 µl of loading buffer, denatured at 95°C for 3 min, and finally loaded onto the sequence gel.
2.2.8 Blotting techniques
2.2.8.1 Dot blotting of DNA onto nitrocellulose filters
Dot-blotting is probably the simplest and least laborious method for monitoring the transgene and hence the routine maintenance of breeding lines. However, dot-blotting does not give any information on the physical integrity of the transgene which must be checked by Southern blotting.
After assembling the dot-blot apparatus according to the manufacturer's instruction, the vacuum pump was switched on, and each well was washed with 20 x SSC. Meanwhile, 10 ug of genomic DNA was denatured at 95°C for 10 min., and placed immediately on ice. To each sample, 150 µl of ice-cold 20 x SSC was added. Then the samples were applied to a separate
well of the dot-blot apparatus. When all the wells were emptied, they were washed with 20 x SSC again. Then the apparatus was dismantled, and the filter was baked for 2 hrs at 80°C.
2.2.8.2 Southern blotting of DNA onto nitrocellulose filters (Southern, 1975)
In the Southern blotting, the transfer of denatured DNA from agarose gels to nitrocellulose membrane is achieved by capillary flow. 20 x SSC buffer, in which nucleic acids are highly soluble, is drawn up through the gel into the nitrocellulose membrane, taking with it the single-stranded DNA which becomes immobilized in the membrane matrix. After electrophoresis of the DNA, the gel was shaken in 0.25 M HCl for the depurination. It was followed by shaking it further in denaturing solution for 30 min, and eventually 45 min in neutralizing solution. The gel was layed on a Whatman filter paper whose ends were in reservoir of 20 x SSC and an equilibrated nitrocellulose filter was placed on the gel. After laying 2 more Whatman filter papers and paper towels, a 500 g weight was also put on the top of the blot. The transfer was carried out overnight. Finally, after disassembling of the blot, the filter was washed shortly in 2 x SSC and the DNA was fixed onto the filter by either baking it at 80°C for 2 hours under vacuum or by UV-crosslinking (120 J; UV Stratalinker 1800, Stratagene, USA).
2.2.8.3 Northern blotting of RNA onto nitrocellulose filters
For the transfer of RNA onto a nitrocellulose filter, the same procedure as above (2.2.6.3) was performed. In this case, however, the gel does not need to be denatured, but is transferred directly onto the filter.
2.2.8.4 Western blotting of protein onto nitrocellulose filters (Gershoni and Palade, 1982)
Towbin Buffer 25 mM Tris pH 8.3
192 mM Glycine
20 % Methanol
After the electrophoresis of proteins on a SDS polyacrylamide gel, the gel and the membrane which was cut at the size of the gel were equilibrated in Towbin buffer for 10 minutes. 2 sheets of Whatman 3MM filter paper were cut and soaked in the transfer buffer too. The gel was placed on these filter papers, and the membrane on the gel avoiding any air bubbles.
Another 2 sheets of filter paper were also wet to complete the sandwich model, and it was placed between the pre-wetted fibre pad, and the cassette was closed and placed into the tank.
The transfer was carried out either at 90 mA at 4°C overnight or at 200 mA at room temperature for 2-3 hours.For the Western blotting of acid-urea gel, the same procedure was employed except from the transfer buffer which was 0.1 M glycine, pH 3.0.
2.2.9 Hybridization of nucleic acids
The membrane to be hybridized was first equilibrated in 2 x SSC, then transferred to a hybridization bottle. After adding 10 ml of hybridization solution, it was incubated for 2 hours in the hybridization oven at an appropriate temperature which was usually 65°C. Then, the labelled probe and salmon DNA were denatured at 95°C for 10 min and added to the hybridization solution. The hybridization was carried out overnight in the oven. Next day, the filter was washed firstly for 10 minutes with 2 x SSC at room temperature, then with 2 x SSC and 0.2 x SSC at the hybridization temperature. When further washing was needed, finally it washed with 0.2 x SSC containing 0.1 % SDS at the hybridization temperature. After drying the filter, it was sealed in Saran wrap, and exposed to an autoradiogram overnight at -80°C.
2.2.10 Isolation of proteins
2.2.10.1 Isolation of total proteins for CAT assay
100 mg of tissue was homogenized in 500 µl of 0.25 M Tris pH 7.8 with a teflon-glass headed pestle. The cell membrane was destroyed by freezing in liquid nitrogen and thawing at 37°C, repeating three times. The samples were centrifuged at 8000 xg for 10 min at 4°C. The proteins which might interfere with the CAT assay were denatured by incubation at 65°C for 10 min. A final centrifugation at 8000 xg for 10 min at 4 °C was carried out, and eventually the supernatant was distributed in several e-cups, frozen in liquid nitrogen, and stored at – 80°C.
2.2.10.2 Isolation of nuclear proteins (Deryckere et al. 1994)
Solution A 0.6 % Nonidet P-40
150 mM NaCl
10 mM HEPES pH 7.9
1 mM EDTA
0.5 mM PMSF (0.5 M stock solution in methanol)
0.5 mM DTT
Solution B 25 % Glycerol
20 mM HEPES pH 7.9
420 mM NaCl
1.2 mM MgCl2
0.2 mM EDTA
0.5 mM DTT
0.5 mM PMSF
2 mM Benzamidine
5 µg/µl Aprotinin
5 µg/µl Leupeptin
5 µg/µl Pepstatin
Between 100 and 500 mg of tissue was homogenized in 15-25 ml of solution A with the use of a 50 ml Dounce tissue homogenizer. It was then centrifuged for 1 minute at 800 xg at 4°C to get rid of any unbroken tissue. The supernatant was centrifuged again for 8 min at 3200 xg.
The pellet which contained nuclei was dissolved in 50-500 µl of Buffer B, and incubated on ice for 20 minutes in order to lyse the nuclei. After a final centrifugation at 14000 xg for 2 minutes, the nuclear protein extract was distributed into tubes and stored at –80°C.
2.2.10.3 Isolation of nuclear basic proteins (Alfonso and Kistler, 1993)
2-4 freshly prepared testis were homogenized in 800 µl of 0.25 M HCl in a 1.5 ml microcentrifuge tube with a teflon-glass headed pestle. Proteins were allowed to extract for 20 min on ice, and 24 µl of TCA was added. The precipatate was removed by centrifugation for 5 min at full speed, and the supernatant was transferred to a new tube. Transition proteins were precipitated by the addition of 200 µl of 100 % TCA, and collected by centrifugation as before for 10 min. The faint precipitate was washed with 700 µl of acetone, and let it dry completely. The pellet then was dissolved in 40-60 µl of 0.5 % acetic acid. This fraction was designated the 3-20 % TCA preparation.
2.2.10.4 Isolation of cytoplasmic S-100 proteins (Dignam et al., 1983, modified)
Buffer A 10 mM HEPES pH 7.9
1.5 mM MgCl2
10 mM KCl
0.5 mM PMSF
0.5 mM DTT
Buffer B 0.3 M HEPES pH 7.9
1.4 M or 2 M KCl 0.3 mM MgCl2
Buffer D 20 mM HEPES pH 7.90.
1 M KCl0.2 mM EDTA
20 % Glycerol
0.5 mM PMSF
0.5 mM DTT
The testes from 5 adult SIV 50 rats were freshly prepared, and shortly washed in cold PBS. 2 testes at a time were homogenized in two Vol of Buffer A by 10 strokes of a B type pestle.
The homogenate was distributed into Eppendorf cups, and centrifuged at 2000 rpm for 10 min at 4°C in order to remove the cell debris. The supernatant was transferred this time to ultracentrifuge e-cups, and a salt shock by adding 0.11 Vol Buffer B was applied. After centrifugation for one hour at 45000 xg at 4°C, the supernatant was collected and dialyzed for 2-4 hrs against 20 Vol of Buffer D. Finally, after a short centrifugation at full speed, the proteins were distributed into several e-cups, freezed in liquid nitrogen, and stored at –80°C.
2.2.11 Determination of protein concentration (Bradford, 1976)
To determine the protein concentration, Bio-Rad protein assay was employed which is a dye binding assay based on the differential color change of a dye in response to various concentrations of protein. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Blue G-250 shifts from 495 to 595 nm when the binding to protein occurs. The BSA stock solution of 1 mg/ml was diluted in order to obtain standart dilutions in the range of 10 µg/ml to 100 µg/ml. The Bio-Rad’s color reagent was diluted 1:5 with H20, and filtered through 0.45 µm filters. In a 96-well microtiter plate, 20 ul of from each standard dilutions and from the samples to be measured were pipetted with 280 µl of the color reagent. The absorption of the color reaction was measured at 595 nm in a microplate reader (Microplate Reader 450, Bio-Rad, Munich).
2.2.12 Reverse transcriptase PCR (RT-PCR)
1-5 ug total RNA was mixed with 1 ul of oligo (dT)18 primer (10 pmol/ul) in a total volume of 11 ul. To avoid the possible secondary structure of the RNA which might interfere with the synthesis, the mixture was heated to 70°C for 10 minutes, and then quickly chilled on ice.
After a brief centrifugation, the followings were added to the mixture:
4 ul 5 x First Strand buffer 2 ul 0.1 M DTT
1 ul 10 mM dNTPs 1 ul Rnasin (10 U/ ul)
The content of the tube was mixed gently and incubated at 42°C for 2 min. Then, 1 ul of reverse transcriptase enzyme (SUPERSCRIPT II RNase H - Reverse Transcriptase, GibcoBRL Life Technologies, USA) was added, and further incubated at the same temperature for 1 hr for the first strand cDNA synthesis. Next, the reaction was inactivated by heating at 70°C for 15 minutes. 10 % of the first strand reaction was used for the PCR reaction.
10 ul 10 x PCR buffer
3 ul 50 mM KCl
2 ul 10 mM dNTP mix 1 ul primer 1 (10 pmol/ ul) 1 ul primer 2 (10 pmol/ ul)
1 ul Taq DNA polymerase (5 U/ul) 2 ul cDNA (from first strand reaction)
80 ul H2O
The reaction was first heated to 94°C for 3 min. for the denaturation of the DNA, then 35 cycles of PCR amplification was performed with appropriate annealing and extension conditions depending on the primers.
2.2.13 Non-radioactive CAT assay
The role of CAT in bacteria is to detoxify the antibiotic chloramphenicol by mono- and diacetylation. CAT activity can therefore be measured by following the conversion of chloramphenicol to its 1-acetyl and 3-acetyl derivates. The mono- and diacetyl derivates of chloramphenicol are separated from unmodified compound by thin layer chromatography on silica gel. Stratagene's Flash CAT nonradioactive CAT assay based on the use of a fluorescent chloramphenicol (CAM) substrate. 50 µg of total protein extract was mixed with 15 µl of BIODIPY CAM substrate reagent and 10 µl of 4 mM acetyl CoA in a total volume of 80 µl, and incubated at 37°C for 4 hrs. Then the reaction was terminated by adding 1.0 ml of ice- cold ethyl acetate. The samples were vortexed and centrifuged at 10000 xg for 5 min. After removing the supernatant, the rest was evaporated for about 90 min. in a vacuum drier. The remaining yellow residue was dissolved in 30 µl of ethyl acetate. Only 5 µl of sample was spotted about 1.5 cm from bottom of the TLC plate. The thin layer chromatography was performed in a closed preequilibrated TLC tank with an 87:13 mixture of chloroform- methanol to depth of 1.0 cm. The result of TLC was visualized under long-wavelength UV light (366-nm).
2.2.14 Primer-extension analysis (Domenjoud, 1990, modified)
Primer extension analysis is used to determine the location of the 5´-end of specific RNAs.
An end-labeled oligonucleotide is hybridized to RNA and is utilized as a primer by reverse transcriptase in the presence of deoxynucleotides. The RNA is thus reverse transcribed into cDNA, which is analyzed on a denaturing polyacrylamide gel. The length of the generated cDNA reflects the number of bases between the labeled nucleotide of the primer and the 5´- end of the RNA.
2.2.14.1 Labelling of the primer
A primer which was estimated to be about 50-200 bp from the transcription start point was labeled with [γ-32P] as follows:
50 pmoles of primer
1.2 µl of Kinase Buffer (10x) 1 µl of T4-Polynucleotide kinase
5 µl of [γ-32P] ATP
in a total volume of 12 µl
The reaction was incubated at 37°C for 45 min, and then stopped with 2 µl of 0.5 M EDTA.
After precipitation, the pellet was dissolved in 15 µl of H2O.
2.2.14.2 Annealing of the primer to RNA
Poly A+-RNA which was isolated from 100 µg of total RNA was mixed with 2-3x106 cpm of labeled primer and 6 µl of hybridization buffer in a total volume of 30 µl. The reaction was immediately placed in a thermoblock which was 85°C for 15 min. It was allowed to cool down till 42°C slowly, and further incubated at this temperature for 2 hrs. Finally, it was precipated by adding 1/10 Vol of 3 M NaAc and 2.5 Vol of 100 % ethanol.
2.2.14.3 Primer extension reaction
To the RNA/oligonucleotide pellet,
12 µl of 5x Reverse transcriptase buffer 12 µl of 5 mM dNTPs
1 µl of RNasin (20 U/ml)
0.5 µl of DTT
2.5 µl Reverse transcriptase (Gibco/BRL) 36 µl H2Owas added.
The reaction was incubated at 42°C for 30 min. Then, the samples were precipitated and dissolved in 10 µl of formamid stopmix.
2.2.14.4 Gel electrophoresis
The samples were denatured at 95°C for 5 min. before they were applied on a 8 % sequencing gel. A sequence reaction of a M13 single-stranded DNA was also run so that the size of the reaction product could be determined. The gel was dried and exposed to an X-ray film.
2.2.15 Analysis of DNA-Protein interaction
2.2.15.1 Electrophoretic mobility shift assay (EMSA)
(Johnson et al., 1991, modified) (Fried and Crothers, 1981; Garner and Revzin, 1981)
The electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-protein binding. The assay is performed by incubating nuclear or cell extract preparations with a 32P end-labeled DNA fragment containing the putative protein binding sites. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The principle of the procedure is that DNA-protein complexes have different mobilities from uncomplexed DNA during PAGE.
Footprinting buffer 24 mM HEPES pH 7.6 80 mM KCl
2 mM EDTA
1.2 mM DTT
16 % Glycerol
To avoid unspecific binding, 10 µg nuclear extract was first incubated with 3 µg poly dI.dC on ice for 15 min. In the case of competition experiments, the unspecific and/or specific competitor DNA was added to the mixture, and further incubated for 15 min. Next, 10000 cpm of labelled oligonucleotide was added and the incubation on ice was continued for 30 min. Finally, the samples were applied to 8 % polyacrylamide gel. After electrophoresis of about 3 hrs, the gel was dried for 45 min and exposed to an autoradiogram overnight at -80°C.
2.2.15.2 Southwestern analysis
(Dai et. al., 1990, Miskimins, et. al., 1985, modified)
Binding Buffer 10 mM HEPES pH 8.0
50 mM NaCl 10 mM MgCl2
0.1 mM EDTA
1 mM DTT
0.25 % Dry milk
40-60 µg of nuclear proteins were separated on 10 % SDS-polyacrylamide gel, and transferred overnight at 4 °C. The filter was incubated in 5 % dry milk in 10 mM HEPES, pH 8.0, for 2 hrs at room temperature. The binding reaction of radioactively labelled oligonucleotides with immobilized proteins was carried out in 15 ml of binding buffer which contains 3 µg of poly dI.dC as the unspecific competitor and 105 cpm of 32P labelled oligonucleotide for 2-3 hrs at room temperature. Finally, the filter was washed two times for 5 min in 10 ml of binding buffer.
2.2.16 Analysis of RNA-Protein interaction
2.2.16.1 RNA-affinity chromatography (Gu et. al., 1996 ; Wu et. al., 1997, modified)
RNA Binding Buffer 25 mM HEPES pH 7.5
100 mM KCl
Washing Buffer A 20 mM HEPES
40 mM KCl 5 mg/ml Heparin 50 µg/ml tRNA
Washing Buffer B 20 mM HEPES
40 mM KCl
0.5 % Nonidet P-40
Washing Buffer C 20 mM HEPES 40 mM KCl
Washing Buffer D 20 mM HEPES
100 mM KCl
Elution Buffers 0.5 M / 1 M / 2 M / 3 M KCl in 20 mM HEPES
Dialyse Buffer 10 mM HEPES
40 mM KCl 3 mM Mg2Cl2 1 mM DTT
0.5 mM PMSF
Polyadenylated RNA transcripts were generated in vitro with the T7 MEGAscript kit (Ambion, USA) from the lineralized plasmid carrying the insert to be transcribed. 500 µl of poly(U)-agarose beads (type 6; Pharmacia) was resuspended in 1 ml RNA binding buffer and packed into a 2-ml column. 40 µg of the in vitro transcribed polyadenylated transcript was added to the column at 4°C and recycled at least 5 times. The efficiency of the binding of RNA to the poly(U)-agarose beads was determined by analyzing the flow-throughs on an agarose gel. Then the column was equilibrated with the protein extraction buffer, followed by addition of about 30-40 mg of testicular cytoplasmic extract. The column was then stored and shaked gently at room temperature for 1 hr to enhance the binding of cytoplasmic proteins to the given transcript. The protein extract contained 60 U/ml RNasin to inhibit endogenous RNase activity, and to minimize the nonspecific binding, heparin and yeast tRNA at final concentrations of 5 mg/ml and 40 µg/ml, respectively, were added. To pellet the agarose beads, the column was centrifuged at 1000 rpm for 5 min. The following steps of washing of the column and elution of the proteins were again carried at 4°C. The column was washed extensively with 2 ml of washing buffer B, buffer C, and finally buffer D. Bound proteins were step eluted with 200 µl of 0.5, 1, 2, and 3 M KCl. The eluted fractions were dialyzed for 2 hrs at 4°C.
2.2.16.2 Northwestern analysis
(Houman et al., 1990; Kwon and Hecht, 1991, modified)
Renaturation Buffer 50 mM Tris, pH 7.6 50 mM NaCl
1 mM EDTA 1 % BSA 1 mM DTT
Binding Buffer 10 mM Tris, pH 7.6
50 mM NaCl 1 mM EDTA 1 mM DTT
After separation of proteins on a 10 % SDS polyacrylamide gel, and transferring onto a nitrocellulose filter, the filter was gently shaked in renaturation buffer at room temperature for 4 hours. It was then shortly equilibrated with the binding buffer, and hybrized with 100000 cpm radioactively labelled in vitro transcript at room temperature for one hour. Finally, the filter was washed 2 times for 10-20 min. again in binding buffer, dried, and exposed to an X- ray film at –70°C overnight.
2.2.17 Immunodetection
AP Buffer 100 mM Tris/HCl pH 9.5
100 mM NaCl
5 mM MgCl2
After Western blotting, the membrane was incubated in 15 ml of 140 mM NaCl, 10 mM Tris/HCl, pH 7.5, 0.05 % Tween containing 5 % Milk powder for 2 hrs at room temperature.
Primary serum which was diluted 1:10000 was added to the mixture and incubation continued for 2 hrs, then the membrane was washed 2 times for 10 min in the same buffer. Upon washing, the blot was incubated with the appropriate alkaline phosphatase-conjugated second antibody which was goat anti-rabbit Ig G in the case of Tnp2 for 2 hrs. The second antibody
was also diluted 1:10000. Finally, the blot was washed again for 10 min. in blocking buffer.
The blot was developed in 10 ml of AP buffer containing 132 ul of NBT 50 mg/ml and 66 ul of BCIP 50 mg/ml.
2.2.17 Histological techniques
2.2.17.1 Tissue preparation for transmission electron microscopy (TEM)
Fixation Solution 0.01 M NaH2PO4.H20
0.04 M Na2HPO4. H20
2.5% Glutaraldehyde
The freshly prepared tissue was incubated in 5 ml of fixation solution overnight at 4°C.
2.2.17.2 Tissue preparation for paraffin-embedding
Bouin’s Solution 15 ml Picrin acid
5 ml 37% Formaldehyde 1 ml Acetic acid
The freshly prepared testis was fixed in Bouin’s solution for 2-4 hrs to prevent the alterations in the cellular structure. The tissue to be embedded in paraffin should be free of water. The dehydration process was accomplished by passing the tissue through a series of increasing alcohol concentrations. For this purpose, the tissue was let in 30%, 70%, 90%, and 100% (2x) ethanol for one hour at room temperature. Later, the alcohol was removed from the tissue by incubating it in methyl benzoat overnight. It was then incubated in 5 ml of roti-clear (Xylol) for 10-30 min at room temperature. The second roti-clear was not discarded but 5 ml of paraplast was added and the incubation was continued at 60°C for another 30 min. The roti- clear and paraffin mixture was discarded, and the tissue was further incubated in 5 ml of paraplast at 60°C overnight. Before embedding, the paraffin was changed at least three times.
Finally, the tissue was placed in embedding mold and melted paraffin was poured into the mold to form a block. The block was allowed to cool and was then ready for sectioning.
2.2.17.3 Peroxidase anti-peroxidase technique (PAP)
Enzyme anti-enyzme soluble complexes have been routinely employed in histochemical techniques. The soluble complex of peroxidase anti-peroxidase (PAP) consists of three peroxidases and two anti-peroxidase subunits in a cyclic structure.The sections were shortly heated to 60°C in order to melt paraffin, and placed in xylol bath for 30 min at 60°C. Before successive ethanol concentrations, they were again incubated in xylol for 30 min at room temperature. Then, the sections were placed in 100% ethanol for 10 min twice, 95%, 80%, 70% ethanol for 5 min. After they were washed in 5-10 min in TBS, the endogenous peroxidase was blocked by the incubation in 3% H2O2 in TBS for 15 min. After washing them once more in TBS for 5-10 min, they were incubated with 7.5 ug Proteinase K for 10 min, and then with 0.1 M Glycin in TBS for 5 min. The incubation with primary antibody which is anti-Tnp2 antibody (1:100 diluted) in our case was accomplished at 37°C for 30 min. After washing shortly with TBS, the sections were this time incubated with the ‘bridge antibody’
for 30 min at 37°C. The incubation with PAP complex (1:150 diluted) was carried out at 37°C for 30 min. The substrate DAB was added to allow the detection of formed complexes.
3. RESULTS
3.1 Transcriptional regulation of rat Tnp2 gene
To investigate the promoter sequence responsible for the testis- and spermatid-specific expression of rat Tnp2 gene, transgenic mice with 147 nts genomic fragment of the 5' regulatory region fused with the E. coli CAT gene as the reporter gene were generated. For the further analysis of this region, in vitro studies such as primer extension analysis and southwestern analysis were carried out.
3.1.1 Primer extension analysis
To determine the transcription start point for rat Tnp2 gene, we carried out primer extension analysis by using a synthetic oligonucleotide with both testis and brain total and poly (A)+ RNA. For the cDNA synthesis, a reverse primer RTP2-PE1 (2.1.5) which is located 78 nts downstream of the translational start codon (ATG) was chosen, and the reaction was carried out on RNA from testis and brain. To estimate the length of the product, a sequencing reaction of M13 phage clone was also run on the gel. The reaction with testis RNA yielded a single product which was 148 nts in length, corresponding to a transcription start site of 70 nts upstream of ATG. As expected, with brain RNA no product was detected as the gene is not expressed in brain but only in testis. A consensus TATA box (TATATAA) could be identified in an expected distance from the transcription start point, which is 24 nts upstream of the transcription start point.
